Recombinant silkworm and silkworm protein comprising heterologous protein produced by the recombinant silkworm

ABSTRACT

The present invention relates to a recombinant organism having any one of nucleic acids (i) to (iv) introduced therein: (i) a nucleic acid having a base sequence of SEQ ID NO: 1; (ii) a nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2; (iii) a nucleic acid encoding a dragline protein and having a sequence identity of 90% or more with the nucleic acid (i); (iv) a nucleic acid which encodes a dragline protein and hybridizes with a complementary chain of the nucleic acid (i) under stringent conditions.

This application is a divisional of U.S. patent application Ser. No.13/226,964 filed Sep. 7, 2011 (now allowed) which claims the benefit ofJapanese Patent Application No. 2010-203558, filed on Sep. 10, 2010, inthe Japanese Patent Office, the disclosures of which are incorporatedherein in their entireties by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a recombinant organism and a proteinproduced by the recombinant organism.

2. Related Background Art

As a natural fiber with an excellent strength, spider silk has drawn anattention. However, spiders eat each other and thus are not suitable forfarming in a same place, and the amount of spider silk obtained from asingle spider is low. In addition, a spider uses different types of silkthreads according to its needs. Therefore, a mass production of spidersilk is difficult.

Then, an attempt has been made to introduce a gene encoding a spidersilk protein into an organism excluding a spider by use of a generecombinant technique to produce spider silk. For example, a method ofproducing a spider silk protein in a genetically modified goat andobtaining the spider silk protein from milk of the goat is disclosed(Patent Literature 1: Japanese Unexamined Patent Application PublicationNo. 2002-506642). However, according to the method, the spider silkprotein needs to be extracted, purified and artificially spun, beingproblematic in the points of labor, cost and environmental load due to asolvent.

To solve the aforementioned problems, an attempt has been made tointroduce a gene encoding a spider silk protein into a silkworm toproduce the spider silk protein (Patent Literature 2: WO2005/068495). Bya recombinant silkworm, the spider silk protein is ejected as a silkthread, and therefore the aforementioned treatments such as extraction,purification and spinning are unnecessary.

SUMMARY OF THE INVENTION

However, physical properties of silk threads produced by conventionalrecombinant silkworms have been far inferior to those of native spidersilks, and the silk threads have been unsatisfactory in their strength.

Then, the present invention aims to provide a recombinant organismproducing a protein with excellent physical properties and a proteinwith excellent physical properties produced by the recombinant organism.Furthermore, the present invention aims to provide a recombinantsilkworm producing a silk thread with a sufficiently excellent strengthand a silk thread with sufficiently excellent strength produced by therecombinant silkworm.

The present inventors intensively studied with a view to achieving theaforementioned aims, and, as a result, found that when a gene of Argiopebruennichi is introduced into an organism to be genetically modified,the resultant protein has excellent physical properties, and therebycompleted the present invention.

More specifically, the present invention relates to a recombinantorganism having any one of the following nucleic acids (i) to (iv)introduced therein and a protein produced by the recombinant organism:

(i) a nucleic acid having a base sequence of SEQ ID NO: 1;

(ii) a nucleic acid encoding a protein having an amino acid sequence ofSEQ ID NO: 2;

(iii) a nucleic acid encoding a dragline protein and having a sequenceidentity of 90% or more with the nucleic acid (i);

(iv) a nucleic acid which encodes a dragline protein and hybridizes witha complementary chain of the nucleic acid (i) under stringentconditions.

By introducing the aforementioned specific nucleic acid, a proteinproduced by the recombinant organism becomes rich in the spider proteinand, as a result, becomes excellent in physical properties such asstrength.

Particularly, the present invention relates to a recombinant silkwormhaving any one of the aforementioned nucleic acids (i) to (iv)introduced therein, a protein produced by the recombinant silkworm and asilk thread produced by the recombinant silkworm. By introducing theaforementioned specific nucleic acid into a silkworm, a silk threadproduced by the recombinant silkworm is rich in spider protein and, as aresult, has a sufficient strength.

According to the present invention, it is possible to provide arecombinant organism which produces a protein with excellent physicalproperties, and a protein with excellent physical properties produced bythe recombinant organism. Furthermore, according to in an embodiment, itis possible to provide a recombinant silkworm which produces a silkthread with a sufficiently excellent strength and a silk thread with asufficiently excellent strength produced by the recombinant silkworm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing the structure of a vector plasmid forgenetic recombination. In FIG. 1, legends have the following meanings:FP: Silkworm fibroin H-chain gene promoter; MASP: Spider gene; FC:C-terminal partial sequence of the silkworm fibroin H-chain gene; MK:Marker gene; L: PiggyBac transposon L-hand; and R: PiggyBac transposonR-hand.

FIG. 2 is a view showing a genome sequence of a silkworm downstream ofthe site at which a spider gene expression cassette is inserted.

FIG. 3 is a view showing the result of the SDS-PAGE separation of silkthread proteins produced by a recombinant silkworm.

FIG. 4 shows scanning electron micrographs of silk threads of arecombinant silkworm (A) and a non-recombinant silkworm (B).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

An embodiment for performing the invention will be described below, ifnecessary, referring to the accompanying drawings. However, the presentinvention is not limited to the following embodiment.

The present invention relates to a recombinant organism having any oneof the following nucleic acids (i) to (iv) introduced therein and aprotein produced by the recombinant organism:

(i) a nucleic acid having a base sequence of SEQ ID NO: 1;

(ii) a nucleic acid encoding a protein having an amino acid sequence ofSEQ ID NO: 2;

(iii) a nucleic acid encoding a dragline protein and having a sequenceidentity of 90% or more with the nucleic acid (i);

(iv) a nucleic acid which encodes a dragline protein and hybridizes witha complementary chain of the nucleic acid (i) under stringentconditions.

Particularly, the present invention relates to a recombinant silkwormhaving any one of the aforementioned nucleic acids (i) to (iv)introduced therein, a protein produced by the recombinant silkworm and asilk thread produced by the recombinant silkworm.

A nucleic acid having the base sequence of SEQ ID NO: 1 is a nucleicacid encoding MaSp1 (major ampullate spidroin 1) protein, which is amain component of a dragline (or warp) protein of Argiope bruennichi,and may be artificially synthesized or obtained from a genomic libraryor a cDNA library, or may be obtained by amplifying each of thesenucleic acids by PCR and obtained by digestion with a restrictionenzyme(s), as long as a nucleic acid has the base sequence of SEQ ID NO:1.

The amino acid sequence of SEQ ID NO: 2 is the amino acid sequence ofthe MaSp1 protein of Argiope bruennichi.

As the nucleic acid to be introduced into a silkworm, a nucleic acid(iii) having a sequence identity of 90% or more with the nucleic acidhaving a base sequence of SEQ ID NO: 1 may be used as long as it encodesa dragline protein. The sequence identity may be 90% or more, but ispreferably 95% or more and more preferably 98% or more.

Furthermore, the nucleic acid to be introduced into a silkworm may be anucleic acid (iv) which hybridizes with a complementary chain of anucleic acid having the base sequence of SEQ ID NO: 1 under stringentconditions as long as the nucleic acid encodes a dragline protein.Herein, “complementary chain” of a nucleic acid refers to a nucleotidesequence which pairs through hydrogen bonding between nucleic acid bases(for example, T to A, C to G). Furthermore, “hybridize” means to form acomplementary bonding between complementary chains or form interactionbetween bases of single-strand nucleic acid molecules.

“Stringent conditions” mentioned above refers to conditions under whicha complementary chain of a nucleotide chain having a homology with atarget sequence preferentially hybridizes with the target sequence and acomplementary chain of a nucleotide chain having no homology does notsubstantially hybridize. The stringent conditions are dependent upon thesequence and vary depending upon various situations. As a sequencebecomes longer, specific hybridization thereof occurs at a furtherhigher temperature. Generally, for stringent conditions, a temperatureis selected so that it is about 5° C. lower than the thermal meltingtemperature (T_(m)) of a specific sequence at a predetermined ionstrength and pH. T_(m) is the temperature at which 50% of complementarynucleotides to a target sequence hybridize with the target sequence inan equilibrium state at a predetermined ion strength, pH and nucleicacid concentration. “Stringent conditions” are dependent upon thesequence and vary depending upon various environmental parameters. Ageneral principle of nucleic acid hybridization can be found in Tijssen(Tijssen (1993), Laboratory Techniques In Biochemistry And MolecularBiology-Hybridization With Nucleic Acid Probes Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assay”, Elsevier, N.Y.).

Typically, the stringent conditions are those in which the saltconcentration is less than about 1.0 M Na⁺, typically about 0.01 to 1.0M of Na⁺ concentration (or another salt) at pH 7.0 to 8.3; and thetemperature is at least about 30° C. for a short nucleotide (forexample, 10 to 50 nucleotides) and at least about 60° C. for a longnucleotide (for example, longer than 50 nucleotides). The stringentconditions can be also achieved by addition of a destabilizing agentsuch as formamide. The stringent conditions referred in thisspecification include hybridization in a buffer solution of 50%formamide, 1M NaCl, 1% SDS (37° C.) and washing with 0.1×SSC at 60° C.

In this specification, the term “recombinant organism” refers to anorganism transformed by introducing a foreign gene into the chromosomeby means of genetic recombination. The organism to be transformed is notparticularly limited and, for example, an insect, an animal, a plant ora microorganism may be used; however, an insect is preferred. Examplesof the preferable insect include Bombyx mori, Bombyx mandarina,Antheraea yamamai and Antheraea pernyi. Among them, Bombyx mori andBombyx mandarina belonging to Bombycidae are preferably used, and Bombyxmori is particularly preferably used.

In this specification, the word “silkworm” refers to Bombyx mori. Asilkworm may be either a breed for experimentation or a commercial breedcommercialized for practical use. Furthermore, the word “recombinantsilkworm” refers to a silkworm transformed by introducing a foreign geneinto the silkworm chromosome by means of genetic recombination. In anembodiment, genetic recombination is performed by a method using atransposon; however, the method is not limited and any method is used aslong as it can introduce a foreign gene into a silkworm andrecombination of a gene can be performed by other methods includingelectroporation.

In this specification, the word “silk thread” is a fiber, which isejected by Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraeapernyi, etc., constituting a cocoon and containing a fibroin protein asa main component. The fibroin protein is composed of two large and smallsubunits (H-chain and L chain).

EXAMPLES

The present invention will be more specifically described by way ofExamples. However, the present invention is not limited to the followingExamples.

(Spider Gene)

A spider gene was obtained in accordance with a PCR method by using avector containing a nucleic acid having the base sequence of SEQ ID NO:1 and primers designed so as to match respectively with two ends of thenucleic acid. To the primers, appropriate restriction enzyme sites arepreviously provided for the following gene manipulation. Morespecifically, as a forward primer, MaSp1FW(5′-CGACTCACTATAGGGAATTCCTTAACTAGTGGAGCAGCC-3′) (SEQ ID NO: 3) was usedand as a reverse primer, MaSp1RV(5′-GACAATCCGTATACCAAGCTTTCTCTGCTAGCTAG-3′) (SEQ ID NO: 4) was used.

(Silkworm Fibroin H-chain Gene Promoter Sequence)

A silkworm fibroin H-chain gene promoter sequence was obtained inaccordance with a PCR method by using primers designed based on thesequence of the silkworm fibroin H-chain gene (GeneBank Registration No.AF226688) and normal silkworm genomic DNA as a template. Morespecifically, as a forward primer, PfibH5′(5′-AAGCTTGTTGTACAAAACTGCC-3′) (SEQ ID NO: 5) containing a HindIIIrestriction enzyme site was used and as a reverse primer, PfibH3′(5′-TGCAGCACTAGTGCTGAAATCGCT-3′) (SEQ ID NO: 6) containing a Spel sitewas used.

(C-terminal Partial Sequence of Silkworm Fibroin H-chain Gene)

A C-terminal partial sequence of the silkworm fibroin H-chain gene wasobtained in accordance with a PCR method by using primers designed basedon the sequence of the silkworm fibroin H-chain gene (GeneBankRegistration No. AF226688) and normal silkworm genomic DNA as atemplate. More specifically, as a forward primer, LBS-FW(5′-CTAGCTAGCAGTTACGGAGCTGGCAGGG-3′) (SEQ ID NO: 7) containing a NheIsite was used and as a reverse primer, LBS-RV(5′-CGGGATCCTAGTACATTCAAATAAAATGCATAC-3′) (SEQ ID NO: 8) containing aBamHI site was used.

(Preparation of Vector Plasmid for Genetic Recombination)

The silkworm fibroin H-chain promoter sequence (FP), a spider genesequence (MASP) and the C-terminal partial sequence of the silkwormfibroin H-chain gene (FC) were sequentially ligated to form a spidergene expression cassette. The spider gene expression cassette wasintroduced into a vector plasmid containing piggyBac transposon toprepare a vector plasmid for genetic recombination. FIG. 1 is aschematic view showing the structure of the vector plasmid for geneticrecombination. The reference symbols of FIG. 1 stand for the followings.

FP: Silkworm fibroin H-chain gene promoter sequence

MASP: Spider gene sequence

FC: C-terminal partial sequence of the silkworm fibroin H-chain gene

MK: Marker gene sequence

L: PiggyBac transposon L-hand

R: PiggyBac transposon R-hand

(Preparation of Recombinant Silkworm)

The vector plasmid for genetic recombination was amplified inEscherichia coli, and purified by “QIAGEN plasmid Midi Kit”(manufactured by QIAGEN) in accordance with the manual attached the kit.A helper plasmid containing a transposase protein gene was also purifiedby the above method. DNA of the plasmids purified above were dissolvedin TE buffer such that the DNA of the vector plasmid for geneticrecombination and the DNA of the helper plasmid were mixed in a ratio of1:1 and precipitated with ethanol. Finally, the concentration of the DNAmixture was adjusted with a phosphate buffer (pH 7) containing 5 mM KClto be 400 ng/ul. This was injected to a silkworm egg of 3-6 hour oldafter egg-laying under microscopic observation.

The egg to which the sample was injected is designated as G0 generation;the G0 generation grew and changed to a moth, which is designated as G0moth; eggs laid by G0 moth as a parent are designated as G1 eggs. G0moth was mated with a moth having no injection and G1 eggs werecollected; and G1 eggs in the incubation state were observed under afluorescent microscope and screened for fluorescence-emitting eggs(positive egg). A population of eggs laid by a single parent moth (G0moth) was treated as a group and the number of groups in which one ormore positive eggs were detected was counted and recorded as the“positive G1 number”. The results are shown in Table 1.

TABLE 1 Number of Number of Number of injection eggs hatched eggsHatching rate Positive G1 (moth) 6426 1200 18.7 12

(Insertion of Spider Gene by Inverse PCR and Confirmation of InsertionSite)

Silkworm genomic DNA was extracted by a known method (see Sambrook andManiatis, Molecular Cloning-A Laboratory Manual). The genomic DNA wascleaved with restriction enzyme HaeIII and thereafter self-ligated.Using this as a template and two pairs of primers respectively matchingwith left and right hands of piggyBac, and a fragment containing asilkworm genome sequence of an insertion site was amplified to identifythe sequence. More specifically, as a left hand forward primer, BacLF(5′-CTTGACCTTGCCACAGAGGACTATTAGAGG-3′) (SEQ ID NO: 9) was used, as aleft hand reverse primer, BacLR (5′-CAGTGACACTTACCGCATTGACAAGCACGC-3′)(SEQ ID NO: 10) was used, as a right hand forward primer,(5′-CCTCGATATACAGACCGATAAAACACATG-3′) (SEQ ID NO: 11) was used, and aright hand reverse primer, (5′-GTCAGTCAGAAACAACTTTGGCACATATC-3′) (SEQ IDNO: 12) was used. FIG. 2 shows a silkworm genome sequence (SEQ ID NO:13) downstream of a site at which a spider gene expression cassette isinserted.

(Protein Analysis of Silk Thread)

A cocoon produced by a recombinant silkworm was excised, and to 1 mg ofcocoon pieces, 50 μl of a 60% LiSCN was added, which was shaken, allowedto stand still at room temperature for 2 hours to solubilize a protein,thereafter, centrifuged at 15000 rpm to remove an unsolved substances,and the supernatant was subjected to SDS-PAGE to separate a spider silkprotein and fibroin of a silk worm. FIG. 3 shows the SDS-PAGE separationresults of the silk thread protein produced by a recombinant silkworm.In FIG. 3, FH stands for a silkworm fibroin H-chain and MASP stands fora spider silk protein. Furthermore, in FIG. 3, in lane 1, an HMWmolecular weight marker was run; in lane 2, the supernatant containing asilk thread protein produced by a recombinant silkworm was run; in lane3, a solution containing 5 times more protein than that in lane 2 wasrun. From the separation results, the content of the spider silk proteinin the silk thread protein was calculated to be 22.5%, which is anumerical value significantly higher than the content of a conventionalrecombinant silkworm.

(Determination of Fineness of Silk Thread under Scanning ElectronMicroscope Observation)

A cocoon produced by a recombinant silkworm was soaked in warm water of40° C. for one minute, fluff was removed, and thread was carefullycollected and acclimated at conditions: 20° C., 65% RH, a day and nightto prepare a sample. The obtained sample was observed by SEM (scanningelectron microscope) S-2380N (manufactured by Hitachi, Ltd.) at avoltage of 10 kV and photographed. Based on the photograph, the diameterand sectional area of a thread were estimated, and based on thesectional area, the strength of the thread was calculated. As a control,a cocoon of a silkworm (non-recombinant silkworm) not geneticallymodified was treated in the same manner. FIG. 4 shows scanning electronmicrographs of silk threads of a recombinant silkworm (A) and anon-recombinant silkworm (B).

(Analysis of Physical Property of Silk Thread)

The cocoon produced by a recombinant silkworm was soaked in warm waterof 40° C. for one minute, fluff was removed, and thread was carefullycollected and acclimated at conditions: 20° C., 65% RH, a day and nightto prepare a sample. The obtained sample was tested for tensile strengthby use of “AUTOGRAPH AGS-J” (manufactured by Shimadzu Corporation) understandard conditions: 20° C., 65% RH. As a control, the silk threadproduced by a silkworm (non-recombinant silkworm) not geneticallymodified was tested for tensile strength in the same manner. As aresult, the strength of the silk thread of a control (non-recombinantsilkworm) was 397.0 MPa (3.49 g/d), whereas the strength of the silkthread produced by the recombinant silkworm was 489.2 MPa (4.27 g/d),which was increased by 22.35%.

On the other hand, in a conventional recombinant silkworm employing agene of e.g., Araneus ventricosus described in Patent Literature 2, thestrength of a control silk thread was 3.78 g/d, whereas the strength ofthe silk thread of a recombinant silkworm was 3.93 g/d, which wasincreased only by 3.96% (see Patent Literature 2, page 29, Table 3).

From the foregoing, in the case of using a specific nucleic acid of thepresent invention, compared to a conventional case of using a gene ofe.g. Araneus ventricosus, the strength of silk threads produced by therecombinant silkworms was found to clearly increase. Table 2 showsphysical properties of the silk thread of the present invention comparedto those of the silk thread of Patent Literature 2.

TABLE 2 Non- recombinant Recombinant Increased rate Content of silkwormsilkworm of strength spider-thread thread (g/d) thread (g/d) (%) protein(%) Patent 3.78 3.93 3.96 5 Literature 2 Present 3.49 4.27 22.35 22.5Invention

A protein produced by the recombinant organism of the present inventionis useful as a naturally occurring material with excellent physicalproperties. Particularly, the silk thread produced by the recombinantsilkworm of the present invention is a naturally occurring material witha sufficiently excellent strength, and thus, the silk thread ispreferably used not only in medical supplies such as surgical suture butalso various uses such as flight equipment, clothing and cosmetics.

1. An isolated nucleic acid selected from the group consisting of (i) anucleic acid having the base sequence of SEQ ID NO: 1; (ii) a nucleicacid encoding a protein having the amino acid sequence of SEQ ID NO: 2;(iii) a nucleic acid with a base sequence identity of 90% or more to thenucleic acid sequence of clause (i); or (iv) a nucleic acid whichencodes a dragline protein and hybridizes with the complementary chainof the nucleic acid of clause (i) under stringent conditions includinghybridization in a buffer solution of 50% formamide, 1M NaCl, 1% SDS at37° C. and washing with 0.1×SSC at 60° C.
 2. A protein encoded by thenucleic acid according to claim
 1. 3. A silk thread comprising theprotein according to claim
 2. 4. A vector comprising the nucleic acidaccording to claim
 1. 5. A method of producing a silk thread accordingto claim 3, comprising: culturing a recombinant silkworm having thenucleic acid according to claim 1 introduced therein, and collecting thesilk thread produced by the recombinant silkworm.